The timing of DNA replication is a critical parameter of cellular growth. It correlates with patters of transcriptional regulation, chromatin modification, chromosome structure and genome evolution. Furthermore, replication timing changes as cells differentiate, and disruption of replication timing correlates with genome instability, suggesting an intimate relation between replication timing and other important aspects of chromosome metabolism. However, the mechanisms that regulate replication timing are still largely mysterious. We have developed, and proposes to test, a detailed, generally-applicable model for the mechanism of replication timing. Our model posits stochastic regulation of origin firing, in which each origin has a characteristic probability of firing, and the average time of origin firing is regulated by that orgin firing probability. We propose that the probability of origin firing is regulated by the number of MCM complexes - the replicative helicase which establishes an origin as a site of replication initiation - loaded during G1. Origins with more MCMs loaded are more likely to fire and thus, on average, fire earlier. Further, we propose that the number of MCMs loaded is regulated by the affinity with which ORC - the MCM loader - binds the origin. Higher affinity origins bind ORC for a greater fraction of G1, thus allowing more MCM complexes to be loaded. Finally, we propose that heterochromatin provides a second level of regulation on top of the MCM-based mechanism of origin timing regulation, such that in heterochromatic regions origin firing is delayed at one or more of the basic steps: ORC binding, MCM loading or MCM activation. We will test our model by mapping ORC binding, MCM binding and origin timing across both the budding and fission yeast genomes using deep-sequencing-based approaches. The complimentary strengths of these evolutionarily distant yeasts allow for a more rigorous test of our model. Furthermore, any mechanisms that are conserved between the two are good candidates for general principles of eukaryotic biology. If our model is confirmed, it will change the way people think about replication timing. Moreover, it will change the direction of the field from a focus on trying to discover the mechanisms of replication timing, to being able to directly test how MCM loading is regulated to control replication timing in metazoan genomes. Furthermore, accurate information about the mechanism of replication timing is essential to understand how replication timing influences the genome reprogramming required for stem-cell maintenance and cellular differentiation, as well as its role in maintaining genome stability and preventing cancer. PUBLIC HEALTH RELEVANCE: Many of the genetic changes that lead to cancer are caused by errors during DNA replication. Proper organization of DNA replication is essential to prevent such errors. The proposed research will elucidate the mechanisms that organize replication, allowing for the identification o new therapeutic targets and diagnostic tools for the treatment and prevention of human cancer.